The genes for histidine utilization (hut) in Klebsiella aerogenes are typical of a large number of genes whose expression is strictly regulated by the available supply of nitrogen. Thus nitrogen regulation of hut serves as a model system for this kind of pleiotropic control. The goal of the research proposed here is to develop an in vitro transcription system for the hut operon of K. aerogenes that is responsive to regulation by the nitrogen regulatory factor(s) and to study the molecular basis for that regulation. The specific objectives of the five year proposal period are: (1) Physical characterization of the regulatory region (hutP) including the DNA sequence of the region and identification of the sites to which regulatory proteins bind; (2) Genetic analysis of the hutP region, including isolation and mapping of hut mutants unable to respond to nitrogen regulation; (3) Characterization of genetic factors required for nitrogen regulation of hutP expression; and (4) Regulation of transcription from hutP in vitro, including regulation by CAP+cAMP and purification of the nitrogen activating protein.